UNIVERSIDAD DE COSTA RICA SISTEMA DE ESTUDIOS DE POSGRADO RELACIONES FILOGENÉTICAS ENTRE INDIVIDUOS DE LA CLASE ASCIDIACEA (TUNICATA) DISTRIBUIDOS EN LA COSTA PACÍFICO NORTE DE COSTA RICA Tesis sometida a la consideración de la Comisión del Programa de Estudios de Posgrado en Biología para optar al grado y título de Maestría Académica en Biología con énfasis en Genética y Biología Molecular MARÍA ISABEL CORDÓN KRUMME Ciudad Universitaria Rodrigo Facio, Costa Rica 2025 ii Dedicatoria Está tesis está dedicada a mis padres Carlos† y Kornelia†, a mis hermanos Carlos, Luis y Eduardo, así como a toda mi familia. Gracias por creer siempre en mí, por su cariño y por ese apoyo incondicional que nunca me ha faltado. No tienen idea de cuánto los quiero ni de lo importante que son y han sido durante esta aventura académica. Gracias por siempre estar. Los quiero muchísimo. En definitiva, no sería la persona que soy hoy sin ustedes y siempre estaré eternamente agradecida. También se la dedico a cada persona que estuvo presente en esta etapa, docentes, colegas, amigos y personal administrativo. A todas esas personas con las que compartí tiempo y espacio a lo largo de esta etapa de aprendizaje, una experiencia realmente inolvidable y que me llevo grabada en el corazón. iii Agradecimientos El presente trabajo fue posible gracias a la Vicerrectoría de Investigación de La Universidad de Costa Rica, por el financiamiento a través del proyecto Relaciones filogenéticas entre individuos de la clase Ascideacea C3099, al proyecto de Biodiversidad marina del área de Conservación Guanacaste, Costa Rica Proyecto BioMarACG B9508 y el apoyo financiero de la DAAD (Deutscher Akademischer Austauschdienst). Expreso mi más profundo agradecimiento al Sistema de Estudios de Posgrado (SEP) por el apoyo económico brindado a través del Programa de Apoyo a Trabajos Finales de Graduación del Fondo Especial de Becas para asistir al Taller de Taxonomía de Ascidias en California, USA. Gracias a este financiamiento, tuve la oportunidad de ampliar mis conocimientos sobre las ascidias, interactuar con expertos en el campo y fortalecer mi formación académica. Este apoyo fue esencial para el desarrollo de mi investigación y me permitió avanzar de manera significativa. Agradezco al Centro de Investigación en Ciencias del Mar y Limnología (CIMAR), UCR, por el apoyo con logística y equipo para desarrollar el trabajo de campo y de laboratorio. Siempre lo recordaré como él lugar en donde excelentes personas comparten una pasión excepcional por el océano, un ambiente que me enseñó y me hizo crecer mucho. A todo el CIMAR, infinitas gracias. A mi comité de tesis, Juan José Alvarado, Melissa Mardones y Laura brenes, por todo su apoyo, confianza, comprensión y paciencia lo largo de esta etapa. A Chepe, mi tutor, por creer en mi desde el inicio y apoyarme a ciegas, y sobre todo por enseñarme esa pasión por la vida submarina, lo espectacular de la vida bajo el agua y enseñarme a ver el mundo diferente. A Melissa por toda la paciencia y el tiempo tomado para enseñarme sobre los análisis bioinformáticos, guiarme y darme buenos consejos. A Laura por el apoyo incondicional, la guía para analizar, procesar y presentar datos, pero también por el tiempo dedicado. A Kaylen por su tiempo de laboratorio, la amistad y todo ese tiempo compartido para ver, estudiar, colectar y hablar de las ascidias. Porque si no hubiera sido por estos misteriosos iv y lindos tunicados, nunca nos hubiéramos conocido. Me llevo buenas memorias y ojalá la vida nos permita seguir esta amistad tan buena que tenemos y nos deje investigar más sobre las ascidias. A Cindy Fernández, quién me abrió las puertas de su laboratorio y me permitió adentrarme en el maravilloso mundo de las algas. Muy agradecida por todo el apoyo y confianza desde el inicio, y por esos momentos de aprendizaje y risas durante giras de campo. A Jeffrey Sibaja, quien siempre estuvo presente y al pendiente, agradezco todo el apoyo brindado, especialmente para la extensión de mi beca y para el procesamiento de las placas en el laboratorio. Asimismo, agradezco a todas las personas que colaboraron en el trabajo de campo Kaylen González, Leonardo Chacón, Arturo Ángulo, Yelba Vega y Gilberth Ampié. No me alcanzan las palabras para agradecer a cada una de esas personas que estuvo presente para que todo fuera exitoso, desde el campo hasta el laboratorio, no me queda nada más que hacerles saber que estoy muy agradecida. Porque sin todos ustedes esta investigación no hubiera sido posible. A mis hermanos, quienes siempre me han apoyado y estuvieron en todo momento. A pesar de la distancia, nunca nos sentimos lejos. Gracias infinitas por no haber dejado de creer en mí y empujarme cada día a ser una mejor persona y profesional. Por toda la comprensión en esos días de silencio, pero sobre todo por su amor. v Hoja de aprobación vi Índice Dedicatoria .................................................................................................................................... ii Agradecimientos ........................................................................................................................... iii Hoja de aprobación ....................................................................................................................... v Lista de cuadros .......................................................................................................................... vii Lista de ilustraciones y figuras .................................................................................................. viii RESUMEN ................................................................................................................................... ix Executive Summary ...................................................................................................................... x Introducción .................................................................................................................................. 1 Objetivos ........................................................................................................................................ 3 Resultados ...................................................................................................................................... 4 Capítulo 1. Ascidian (subphylum Tunicata: Class Ascidiacea) species reassessment in the North Pacific of Costa Rica: new records and status evaluation ............................................... 6 Capítulo 2. Want to know about a relative of ours? Phylogenetic relationships among solitary and colonial ascidians (subphylum Tunicata; class Ascidiacea) from the northern Pacific of Costa Rica ................................................................................................................... 38 Conclusión ................................................................................................................................... 58 Recomendaciones ........................................................................................................................ 59 Referencias .................................................................................................................................. 60 vii Lista de cuadros Table 1. Species collected and Status in the North Pacific from Costa Rica. ................... 12 Table 2. Species whose mtCOI sequences were used the phylogenetic analysis of this study. .................................................................................................................................. 43 Table 3. Nucleotide and Haplotypic diversity among Ascidiacea Orders ........................ 46 Table S 1. Species Collection Overview by Sampling Site ........................................................... 57 Table S 2. Summary of Species Collected by Site, Depth, and Taxonomic Order......................... 57 Table S 3. Evaluated Morphological Characters for Species Identification .................................. 57 Table S 4. BLAST Analysis of Obtained 18s rRNA Sequences .................................................... 57 Table S 5. BLAST Analysis of Obtained Coi Sequences .............................................................. 57 Table S 6. Accession Numbers of Obtained and Analyzed Sequences .......................................... 57 viii Lista de ilustraciones y figuras Figure 1. Sampling sites (black triangles) along the North pacific of Costa Rica. ...................... 10 Figure 2. Specimens collected in the Northern Pacific of Costa Rica. ........................................ 14 Figure 3.Consensus tree obtained using Maximum likelihood tree (ML) derived from the Coi dataset. ................................................................................................................................... 47 Figure 4. Maximum likelihood tree (ML) derived from the 18s RNA dataset. ........................... 48 Figure 5. Phylogenetic relationship of colonial and solitary ascidians inferred from the complete Coi dataset. .................................................................................................................... 49 Figure 6. Phylogeny of tunicates inferred from the complete 18s rRNA dataset. ....................... 50 ix RESUMEN La clase Ascidiacea, perteneciente al filo Chordata y subfilo Urochordata, agrupa organismos exclusivamente marinos, sésiles y hermafroditas, con cuerpos de formas irregulares. Es el grupo más diverso del filo Tunicata, desempeñan roles ecológicos clave como filtradores y bioindicadores ambientales. Además, su relevancia se extiende al ámbito evolutivo debido a su cercana relación filogenética con los vertebrados. La identificación morfológica de ascidias enfrenta desafíos debido a su complejidad estructural, lo que ha fomentado el uso creciente de herramientas moleculares para la identificación de especies y elucidar sus relaciones filogenéticas. Sin embargo, en Centroamérica, particularmente en Costa Rica, los estudios tanto morfológicos como moleculares de este grupo son limitados. Este estudio tuvo como objetivo identificar morfológica y molecularmente las especies de la clase Ascidiacea presentes en el Pacífico Norte de Costa Rica y reconstruir sus relaciones filogenéticas mediante inferencia bayesiana y máxima verosimilitud, empleando los marcadores moleculares mtCOI y 18s rRNA. Entre 2019 y 2024, se recolectaron 456 especímenes en 32 sitios de muestreo ubicados en la costa norte del Pacífico costarricense. Estos especímenes representan 33 especies distribuidas en 16 géneros, siete familias y tres órdenes. Los resultados revelaron agrupamientos filogenéticos estables dentro de la clase Ascidiacea, en su mayoría consistentes con la clasificación taxonómica actual. El marcador mtCOI mostró una mayor resolución taxonómica en comparación con el 18s rRNA. Este estudio contribuye significativamente al conocimiento de la diversidad de ascidias en la región, resaltando el valor de las herramientas moleculares para abordar los vacíos existentes en su taxonomía y filogenia. Ingresar al siguiente enlace: https://issuu.com/ascidiasmick/docs/bookletpdf2 https://issuu.com/ascidiasmick/docs/bookletpdf2 x Executive Summary The class Ascidiacea, belonging to the phylum Chordata and subphylum Urochordata, consists of exclusively marine, sessile, and hermaphroditic organisms with irregularly shaped bodies. This group is the most diverse in the phylum Tunicata and plays key ecological roles as filter feeders and environmental bioindicators. Additionally, their evolutionary significance is underscored by their close phylogenetic relationship with vertebrates. Morphological identification of ascidians is challenging due to their structural complexity, which has led to an increasing reliance on molecular tools for species identification and the elucidation of their phylogenetic relationships. However, in Central America, particularly in Costa Rica, there is a limited body of morphological and molecular studies on this group. This study aimed to identify morphologically and molecularly the species of Ascidiacea present in the North Pacific of Costa Rica and to reconstruct their phylogenetic relationships using Bayesian inference and maximum likelihood, with mtCOI and 18s rRNA molecular markers. Between 2019 and 2024, 456 specimens were collected from 32 sampling sites along Costa Rica's northern Pacific coast. These specimens represent 33 species distributed across 16 genera, seven families, and three orders. The results revealed stable phylogenetic groupings within Ascidiacea, largely consistent with current taxonomic classification. The mtCOI marker provided higher taxonomic resolution than 18s rRNA. This study significantly enhances our understanding of ascidian diversity in the region and highlights the importance of molecular tools to address gaps in their taxonomy and phylogeny. 1 Introducción La clase Ascidiacea pertenece al subfilo Urochordata dentro del filo Chordata (Satoh et al., 2014). Estos organismos se caracterizan por poseer varias características básicas de los cordados (cola postanal, cordón nervioso dorsal, notocorda, hendiduras branquiales y endostilo). Sin embargo, carecen de columna vertebral y en cambio, presentan notocorda solo en las etapas larvales (Bastida-Zavala et al., 2014). Son un grupo exclusivamente marino, bentónico, sésil, hermafrodita y con morfología corporal esférica e irregular (Monniot et al., 1991). La clase Ascidiacea (del griego “saco pequeño”) es el grupo más diverso del subfilo Tunicata (o Urochordata), con aproximadamente 3,000 especies divididas en tres órdenes: Aplousobranchia, Stolidobranchia y Phlebobranchia. Se han descrito tres tipos de organización de vida: solitarias, coloniales y mixtas (Lahille, 1886; Shenkar & Swalla, 2011; González-Sánchez et al., 2021). El plan corporal básico de esta clase está compuesto por tres capas dispuestas una dentro de la otra. La capa externa conocida como túnica, compuesta de celulosa, la capa intermedia llamada manto, y la capa interna que comprende el saco branquial. Estas capas presentan dos aberturas: el sifón oral, por donde ingresa el alimento; y el sifón anal, por donde expulsan los residuos de la digestión y los gametos (Monniot & Monniot, 1978). Las ascidias se alimentan por filtración activa, fundamentalmente de plancton (Petersen, 2007). El interés en el estudio de ascidias ha incrementado recientemente, particularmente en los procesos evolutivos, debido a su estrecha relación filogenética con los vertebrados (Cleto et al., 2003; Lemaire et al., 2008). Según datos morfológicos, se ha determinado que los tunicados son el grupo hermano más cercano de los cordados (Bourlat et al., 2006; Delsuc et al., 2006). No obstante, estudios filogenéticos han desarrollado distintas hipótesis debido a la inestabilidad de su posición en el árbol filogenético, dependiendo de los genes utilizados. Zeng & Swalla (2005) identificaron a la clase Ascidiacea como un grupo monofilético, debido a sus características únicas, a pesar de la diversidad morfológica, de ciclos de vida y métodos de reproducción. Recientemente, se ha demostrado que la clase Ascidiacea es parafilética, y que las familias Phlebobranchia y Aplousobranchia están 2 más relacionadas con Thaliacea que con Stolidobranchia (Delsuc et al., 2018). Aunque la posición de Thaliacea no siempre estuvo respaldada filogeneticamente, siempre apareció como grupo hermano de Phlebobranchia y Aplousobranchia, sugiriendo que evolucionaron a partir de un ancestro sésil común (Delsuc et al., 2018). Se destaca la necesidad de una revisión taxonómica exhaustiva para mejorar los esquemas de clasificación, dado que se observa monofilia en el clado que une a Phlebobranchia y Aplousobranchia, lo que sugiere reutilizar el término Enterogona (Garstang, 1928; Delsuc et al., 2018). Un estudio utilizando secuencias del gen nuclear 18s rRNA determinó que Thaliacea es un grupo monofilético y hermano de Aplousobranchia, y sugiere que la colonialidad dentro de este orden se desarrolló independientemente (Turon & López- Legentil, 2004; Piette & Lemaire, 2015). En la región centroamericana, especialmente en Costa Rica, los estudios sobre la identificación morfológica y molecular de las ascidias son escasos (Tokioka, 1972; Gonzalez-Pestana et al., 2017; Roth et al., 2017; Nova-Bustos et al., 2010; Nova Bustos et al., 2016). En Costa Rica, se ha determinado que las ascidias destacan entre los miembros de la fauna bentónica de los fondos rocosos, con ocho especies registradas, correspondientes a cinco géneros, cuatro familias y tres órdenes dentro de Ascidiacea (Tokioka, 1972; González-Sánchez et al., 2021). La falta de investigaciones sobre este grupo se ha atribuido a la dificultad en la identificación taxonómica, ya que muchas estructuras diagnósticas son difíciles de observar y requieren disecciones especializadas (Stefaniak, 2009). Este estudio permitirá dilucidar las relaciones filogenéticas de las especies de Ascidiacea presentes en Costa Rica, aportando identificación morfológica, molecular e información sobre las relaciones entre ellas. Además, generará un listado actualizado de las especies presentes en el Pacífico Norte de Costa Rica, lo que facilitará estudios adicionales sobre su ecología, interacciones en el ecosistema y distribución. 3 Objetivos A. General Caracterizar morfológica y molecularmente las especies presentes de Ascidiacea distribuidas en la Costa Norte Pacífica de Costa Rica mediante el uso de marcadores moleculares y estandarización de protocolos para su identificación. B. Específicos 1. Reconstruir las relaciones filogenéticas de las especies de Ascidiacea por medio de inferencia bayesiana y máxima verosimilitud con los marcadores moleculares mtCOI y 18s rRNA y Ant. 2. Estimar la diversidad genética y las diferencias genéticas entre las especies de Ascidiacea presentes en el Pacífico Norte de Costa Rica. 3. Describir las características morfológicas diagnosticas de las especies de ascidias presentes en el Pacífico Norte de Costa Rica para estandarizar un protocolo de identificación morfológica. 4 Resultados En total, se recolectaron 456 especímenes de ascidias durante 15 muestreos realizados en 32 sitios de la costa norte del Pacífico de Costa Rica, siendo 51% del orden Stolidobranchia, 33% de Aplousobranchia y 16% de Phlebobranchia (Tabla S1). Se identificaron morfológicamente 33 especies correspondientes a 16 géneros, siete familias (Ascidiidae, Diazonidae, Didemnidae, Polycitoridae, Polyclinidae, Pyuridae y Styelidae), y tres órdenes dentro de la clase Ascidiacea (Tabla 1, S3). Entre los sitios, Pochote (PO) destacó por ser el más diverso, con 13 especies, seguido por Pithaya (PT), Meros (MER) y Refugio (RE), con 12 especies cada uno, y Bajo Viejon (BV) con 10 especies. La mayoría de los sitios mostró representaciones de especies de los tres órdenes de ascidias (Tabla S2). Las especies más comúnmente presentes en estos cinco sitios, con profundidades que oscilan entre 6 y 12.3 metros, fueron Ascidia sideralis, Didemnum cf. perlucidum, Pyura carmanae, Pyura cf. imesa, Rophalaea birkelandi y Symplegma brakenhielmi. Las especies más abundantes fueron P. carmanae, presente en 24 de los 32 sitios, seguida por R. birkelandi en 20 sitios y A. sideralis en 16 sitios. De las 17 especies previamente reportadas, se encontraron 15, y se incluyeron ocho nuevos registros para la costa norte de Costa Rica: Aplidium cf. glabrum, Ascidia cf. munda, Ascidia cf. paratropa, Hermania cf. fimbriae, Pyura cf. imesa, Botrylloides cf. nigrum, Metandrocarpa taylori, Polyandrocarpa cf. zorritensis y Symplegma brakenhielmi. Se generó una guía de campo que incluye todas las especies documentadas, destacando sus principales características morfológicas y su clasificación (Field Guide Costa Rica Ascidians, PDF) Se obtuvieron un total de 224 secuencias de especies de tunicados previamente no muestreadas, de las cuales 68 correspondieron al gen 18s rRNA y 156 al gen mtCOI. El conjunto de datos del gen mtCOI incluyó 156 secuencias recién generadas, 14 secuencias obtenidas de Gene Bank y un Outgroup (Branchiostoma floridae). El conjunto de datos de 18s rRNA incluyó 68 secuencias nuevas y un Outgroup (Branchiostoma floridae). Los árboles filogenéticos obtenidos con los algoritmos de máxima verosimilitud (ML) e inferencia bayesiana (BI) para mtCOI y 18s rRNA respectivamente, mostraron un fuerte 5 apoyo para los principales órdenes taxonómicos, con los valores de bootstrap y las probabilidades posteriores bayesianas correspondientes en los nodos. La selección del modelo inteligente (SMS) basada en el Criterio de Información de Akaike (AIC) mostró que el modelo GTR + G fue el que mejor se ajustó entre los evaluados. Las topologías de los árboles apoyaron la clasificación filogenética más precisa obtenida para los conjuntos de datos de cada gen, mostrando que el conjunto de datos de mtCOI se divide en tres grandes clados, correspondientes a cada uno de los órdenes. El orden Aplousobranchia aparece como un grupo parafilético, mientras que los órdenes Phlebobranchia y Stolidobranchia se presentan como monofiléticos. El conjunto de datos de 18s rRNA se divide en dos grupos, correspondientes a los grupos previamente conocidos Pleurogona y Enterogona. Para un análisis más detallado, se excluyó el orden Aplousobranchia en el análisis de 18s rRNA debido a las posiciones inestables de las secuencias, especialmente para las secuencias de Rophalaea birkelandi. Se realizó un análisis específico para Aplousobranchia por separado debido a esta inestabilidad en las posiciones de las secuencias. 6 Capítulo 1. Ascidian (subphylum Tunicata: Class Ascidiacea) species reassessment in the North Pacific of Costa Rica: new records and status evaluation María Isabel Cordón-Krumme 1,2 https://orcid.org/0000-0001-5974-5865 Melissa Mardones-Hidalgo 2 https://orcid.org/0000-0002-4402-7817 Laura Brenes-Guillén 2,4 https://orcid.org/0000-0002-7185-4084 Kaylen González-Sánchez 1,4 http://orcid.org/0000-0002-7208-9302 Juan-José Alvarado 1,2,3 http://orcid.org/0000-0002-2620-9115 1 Centro de Investigación en Ciencias del Mar y Limología (CIMAR), Universidad de Costa Rica, 11501- 2060, San José, Costa Rica 2 Escuela de Biología, Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 3 Museo de Zoología, Centro de Investigación en Biodiversidad y Ecología Tropical (CIBET), Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 4 Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, 11501- 2060, San José, Costa Rica Abstract | The class Ascidiacea has around 3000 described species, classified into three orders (Aplousobranchia, Stolidobranchia, and Phlebobranchia). Despite their importance and presence, the Costa Rican ascidian fauna is understudied. This study aims to study ascidian diversity in the northern Pacific coast of Costa Rica by characterizing species morphologically, contributing to knowledge of marine biodiversity. Between 2019 and 2024, 456 ascidian specimens were collected, resulting in the identification of 33 ascidian species belonging to16 genera, seven families, and three orders (54% Stolidobranchs, 31% Aplousobranchs, and 15% Phlebobranchs). The species were classified as native, introduced, or cryptogenic, using established criteria to reflect their ecological status. The study highlights the biodiversity of ascidians from shallow waters in the region, with notable concerns about the presence of introduced species such as Polyclinum constellatum and Didemnum cf. perlucidum, which pose potential threats to native marine communities by competing for resources and altering habitats. The presence of cryptogenic species and difficulties in species identification indicate a need for further taxonomic and genetic studies to clarify species status. https://orcid.org/0000-0001-5974-5865 https://orcid.org/0000-0002-4402-7817 https://orcid.org/0000-0002-7185-4084 http://orcid.org/0000-0002-7208-9302 http://orcid.org/0000-0002-2620-9115 7 Introduction The class Ascidiacea, commonly known as sea squirts, represents a significant component of marine biodiversity within the subphylum Tunicata. Ascidians exhibit key chordate characteristics, including the presence of a postanal tail, a dorsal nerve cord, a notochord, gill slits, and an endostyle (Monniot et al., 1991). However, unlike typical chordates, they lack a backbone and possess a notochord and a postanal tail exclusively during the larval stage (Monniot et al., 1991; Corbo et al., 2001; Bastida-Zavala et al., 2014). Comprising approximately 3000 species (Shenkar & Swalla, 2011), ascidians are notable for their tunics composed of cellulose and a special arrangement of cells making it a mesenchyme- like tissue (Smith & Dehnel, 1971; Hirose, 2009). The classification of ascidians is based on the structure of their branchial sac, which has led to the establishment of three orders: Aplousobranchia, Stolidobranchia, and Phlebobranchia (Lahille, 1886). These exclusively marine, benthic, sessile, and hermaphroditic animals exhibit an irregular spherical body morphology (Monniot et al., 1991). Ascidians can be solitary, colonial, or social and are found in diverse marine environments, from shallow intertidal zones to the ocean depths (Shenkar & Swalla, 2011). They feed primarily through filtration of organic matter and plankton (Lambert, 2005; Petersen, 2007). Ascidians are frequently identified as introduced species, primarily transported via ship hulls and ballast water (Lambert, 2002, 2007; López-Legentil et al., 2015; Nydam et al., 2022). With approximately 90% of global trade relying on ships, the transfer of organisms between coastal waters through biofouling and ballast water is widespread (Zhang et al., 2020). Ocean warming further accelerates this issue, as it is expected to hasten recruitment, enhance growth rates, and support the spread of introduced ascidians, potentially altering benthic community structures (Stachowicz et al., 2002; Sorte et al., 2010; Lyman et al., 2010). Many ascidians are invasive, disrupting marine ecosystems, biodiversity, aquaculture, and fisheries due to their competitive edge, rapid growth, and environmental tolerance (Shenkar & Swalla, 2011; Aldred & Clare, 2014; Zhang et al., 2020). This resilience underscores the need for detailed taxonomic studies to monitor their impact. 8 The status evaluation of ascidian species into native, introduced, or cryptogenic categories is crucial for understanding their ecological roles and impacts on marine ecosystems (Carman et al., 2011; Lambert, 2019). Native species are autochthonous to the region, while introduced species are those brought in by human activities, such as shipping and aquaculture, often becoming invasive and threatening local biodiversity (Carlton, 1996; Lambert, 2007). Cryptogenic species, which are neither clearly native nor introduced, pose additional challenges in classification due to unclear origins (Carlton, 1996). In the context of Costa Rica, where ascidian biodiversity remains largely unexplored and historical taxonomic studies are scarce, applying these categories requires overcoming significant gaps in baseline data. Determining the status of each species requires a multifaceted approach. An approach that includes morphological and molecular analysis and if available historical data (Carman et al., 2011; Lambert, 2019). These methods help differentiate species and assess their ecological impact, which is essential for effective management and conservation strategies. They are integral components of benthic communities, contributing to the structural complexity of marine habitats (Palomino-Alvarez et al., 2019). The North Pacific is known for its high species diversity, attributed to the unique ecosystems and the influence of upwelling events (Cortés, 2017; Cortés & Joyce, 2020; Fernández-García et al., 2021). Tokioka (1972) was the first to report on ascidian fauna in Costa Rica. He established the presence of 14 species along the Pacific coast and highlighted the importance of showing the species identification distributed across the isthmus due to difficulties in the dissection process. Later Nova-Bustos et al. (2010) reported the presence of five species in three sites within Santa Elena Bay, showing high abundance of Rophalaea. birkelandi and Ascidia certaodes, suggesting a strong relationship with the upwelling event. The most recent study on ascidians in the northern region establishes the presence of eight species (González-Sánchez et al. 2021), underlining the necessity of new sampling efforts to increase our ascidian diversity knowledge. 9 Despite their recognized prevalence in the North Pacific of Costa Rica, ascidian taxonomy and ecology in this region have been poorly studied. The objective of this study is to assess ascidian diversity in the Northern Pacific of Costa Rica using a morphological approach. Given the country's rich marine biodiversity and its position as a biological corridor, understanding ascidian diversity is essential for monitoring invasive species, preserving local ecosystems, and contributing to the broader knowledge of Costa Rican marine fauna. Materials and Methods Study area: First surveys were done in 2018, 2019 and 2021, one each year. The latest sampling occurred during four field expeditions of four days during April, August and November 2023 and July 2024. The sampling sites were located at 31 different locations within the Guanacaste Conservation area (ACG, Área de Conservación de Guanacaste) in Costa Rica’s North Pacific region (Fig. 1). The coastal environments of the northern Pacific of Costa Rica, which includes ACG encompass a range of characteristics landforms, including rocky shores, bays, islets and beaches, which are distributed across Bahía Santa Elena and Islas Murciélago. It is known for being a relatively shallow bay having 35 m deep at external zones, having sandy and rocky bottoms made up of columnar and massive basalts (Cortés, 2016; Fernández-García et al., 2021). This area experiences a seasonal upwelling from December to April-May, bringing up cooler, nutrient-rich waters and maintaining an average surface seawater temperature of approximately 22°C (Jiménez, 2001; Alfaro et al., 2012; Lizano & Alfaro, 2014). 10 Figure 1. Sampling sites (black triangles) along the North pacific of Costa Rica. Sampling sites: Barco hundido (BH), Bajo Junquillal (BJ), Bajo la vita (BLV), Bajo Rojo (BR), Bajo Viejón (BV), Colorado Sur (CS), Casa Verde Bajo Chaca (CVB), Isla David (ID), Isla Cocinera (IC), Islita (IS), Jardín (JA), La Cornuda (LAC), Los Cabros (LC), La mesa (LM), Loros (LO), Meros (MER), Mango (MG), Muñecos (MÑ), Mogotes (MO), Montosa (MON), Matapalito (MT), MyM (MyM), Palmares (PAL), Isla Pelonas (PEL), Puerta de la Iglesia (PI), Pochote (PO), Isla Playa Rajada (PR), Pithaya (PT), Refugio (RE), Bahía Thomás (TB), Virador (VR). Sampling: The sampling targeted colonial and solitary organisms at depths ranging from 1 to 17 meters on rocky bottoms and reef environments using SCUBA gear. The specimens were extracted with the utmost care using a metal spatula and/or a sharp knife. They were then transported to the surface in plastic bags containing seawater. Sample processing: All the collected specimens were placed inside a plastic bag with seawater and menthol crystals for relaxation and were preserved in formalin at 4% (Shenkar & Loya, 2009; Rocha & Counts, 2019). The duration of this process varied among the orders and the size of the specimens, the order Stolidobranchia typically 11 requiring several hours. To ascertain whether the specimen has reached the requisite level of relaxation, a fine stick was used to approach the siphons and observe for movement. In the absence of such movement, the identification process was then be initiated. Taxonomic identification: Each collected specimen was photographed, For most cases it was necessary to carry out external and internal dissection, following routine methods (Monniot & Monniot, 1972) and the literature for Pacific ascidians and adjacent regions was consulted for identification (Michaelsen, 1904; Van Name, 1931, 1945; Tokioka, 1971, 1972; Kott & Harris, 1972; Kott, 1985, 1990; Monniot et al., 1991; Kott, 2002; Lambert, 2002; Carman et al., 2011; Bullard et al., 2011; Rocha et al., 2012; Bonnet et al., 2013; Rocha & Counts, 2019; Lambert, 2019). All the collected specimens were deposited at the Museum of Zoology, Centro de Biodiversidad y Ecología Tropical (CIBET), Universidad de Costa Rica. Results A total of 456 ascidian specimens were collected during the 15 surveys along 32 samplings sites on the northern Pacific coast of Costa Rica, being 51% Stolidobranchs, 33 % Aplousobranchs and 16% Phlebobranchs (Table S1). A total of 33 species were morphologically identified, corresponding to 16 genera, seven families (Ascidiidae, Diazonidae, Didemnidae, Polycitoridae, Polyclinidae, Pyuridae and Styelidae), and three orders within class Ascidiacea (Table. 1, S3). Among all sites Pochote (PO) was the most diverse with 13 species, followed by Pithaya (PT), Meros (MER), and Refugio (RE) with 12 species each, and Bajo Viejon (BV) with 10 species. Most of the sites showed representations of species from the three ascidian orders (Table S2). The species consistently present across these five sites ranging in depth from 6 to 12.3 m were Ascidia sideralis, Didemnum cf. perlucidum, Pyura. carmanae, Pyura. cf. imesa, Rophalaea birkelandi, and Symplegma brakenhielmi. The most abundant species identified were P. carmanae, found at 24 of the 32 sites followed by R. birkelandi at 20 sites, and A. sideralis, present at 16 sites. We found 15 of the 17 species previously reported and we include eight new records for the northern coast of Costa Rica: Aplidium cf. glabrum, Ascidia cf. munda, Ascidia cf. paratropa, Hermania cf. fimbriae, Pyura cf. imesa, 12 Botrylloides cf. nigrum, Metandrocarpa taylori, Polyandrocarpa cf. zorritensis and Symplegma brakenhielmi. Species status evaluation Each species in the present study was assigned a status of native, introduced, or cryptogenic based on the criteria outlined by Chapman and Carlton (1991) and Carlton (1996). For further details on the criteria used for status determination, see the methodologies previously applied by Carman et al. (2011) and Lambert (2019). Table 1. Species collected and Status in the North Pacific from Costa Rica. I: Introduced; C: Cryptogenic and N: Native. * Not evaluated recently. Species Status Site(s) in this study Other studies Aplousobranchia Diazonidae Rhopalaea birkelandi N Bajo Viejón, Muñecos, Mogotes + 17 Costa Rica (Tokioka, 1972; Carman et al., 2011; González-Sánchez et al., 2021) Didemnidae Didemnum cf. perlucidum I Bajo Viejón, Meros, Pochote + 3 Pacific Panamá (Carman et al., 2011), Galápagos (Lambert, 2019) Didemnum spp. n/a Montosa, Isla David, Viardor + 4 Diplosoma cf. listerianum I Bahía Thomas, Refguio + 1 Reported Cryptogenic for Pacific Panamá by (Carman et al., 2011), Galápagos (Lambert, 2019). Diplosoma sp. n/a Pochote y Refgio Lissoclinum cf. fragile C Montosa y Refugio Polysyncraton sp. n/a La Cornuda, Isla Playa Rajada Polycitoridae Cystodytes cf. dellechiajei C Mogotes, Isla Playa Rajada, La Mesa + 8 Paraná, Brasil(Rocha & Kremer, 2005), Galápagos (Lambert, 2019) Cystodytes sp. n/a Mango y Mogotes Polyclinidae Aplidium cf. glabrum C Pochote Aplidium cf. stellatum N Isla Cocinera, Barco Hundido, Muñecos + 1 Polyclinum constellatum I Bahía Thomas Galápagos (Lambert, 2019) Aplousobrachia_ indet. 12 13 Phlebobranchia Ascidiidae Ascidia ceratodes N Bajo Rojo (Carman et al., 2011), Reported as Cryptogenic in Galapagos Islands (Lambert, 2019) Ascidia cf. munda N* Montosa, Loros, La Cornuda + 1 Western Australia (Kott, 1985), Distributed along the Central Indo-Pacific (Van der Land, 1994). Ascidia cf. paratropa N* Islita North East Pacific (Van der Land, 1994) Ascidia sideralis N Colorado Sur, Pochote, Mando +13 Recently described for Pacific Panama (Bonnet et al., 2013) Ascidia spp. n/a 9 Ascidia sydneiensis I Bajo Junquillal, Pithaya, La Cornuda + 3 Pacific and Atlantic Panamá (Carman et al., 2011)Galápagos (Lambert, 2019) Phlebobranchia_ indet. Isla David Stolidobranchia Pyuridae Herdmania sp. n/a Meros Herdmania cf. fimbriae I Matapalo Herdmannia momus reported for Indo Pacific (Rocha et al., 2012) Microcosmus exasperatus C Barco hundido, Bahía Thomas, Pithaya + 3 Pacific Panamá and Indo Pacific (Carman et al., 2011; Rocha et al., 2012) and reported introduced in Galapagos Islands (Lambert, 2019) Pyura carmanae N Bajo la Vita, La Cornuda, Pochote + 21 Recently described for Panamá Pacific (Rocha & Counts, 2019) Pyura cf. imesa N Pithaya, Meros, Mango + 11 Recently described for Pacific Panamá (Rocha & Counts, 2019) Pyura lignosa N Palamares, La Cordnuda, Los Cabros + 9 (Van Name, 1945; Monniot, 1994; Carman et al., 2011) Pyura sp. n/a Los Cabros y Muñecos Pyura stolonifera I Pithaya, Pochote, La Mesa + 5 Perú and Ecuador (Van Name, 1931), Costa Rica (González-Sánchez et al., 2021) Styelidae Botrylloides cf. nigrum I Bajo Viejón, Islas Pelonas, La Cornuda + 1 C for Pacific Panamá (Carman et al., 2011), (Lambert, 2019) Botrylloides spp. n/a Barco hundido, Pochote + 4 Metandrocarpa taylori N Bajo Viejón, La Mesa, +4 North Pacific Ocean (Van der Land, 1994) Polyandrocarpa cf. zorritensis I Isal Cocinera + Meros Pacific Panamá (Carman et al., 2011), Galápagos (Lambert, 2019) Polyandrocarpa spp. n/a Barco hundido, Jardín, Palmares + 6 Polyandrocarpa anguinea C Pithaya (2019) Atlantic Panamá (Carman et al., 2011) 14 Symplegma brakenhielmi I Bajo Vejó, Bajo Junquillall, Mogotes + 3 Symplegma sp. n/a 3 Reported native for the Caribbean (Streit et al., 2021) Stolidobranchia_ indet. 2 Systematics In this section, we provide a brief description of the 33 species collected during sampling along the north Pacific coast and within the ACG (Table S3). The descriptions focus primarily on the most distinctive characteristics of the species, particularly on key structures. Any characteristics not mentioned in the description are assumed to match the original species description. Figure 2. Specimens collected in the Northern Pacific of Costa Rica. Each color represents the order to which the specimens belong, Purple= Aplousobranchia, Green= Phlebobranchia and Orange= Stolidobranchia. A.Rhopalaea birkelandi, purple and pink specimens with tunic; B. Polyclinum constellatum, whole colony; C. Didemnum perlucidum., whole colony; D. Cystodytes sp.,whole colony; E. Aplidium cf. glabrum, whole colony and extracted zooids; F. Diplosoma listerianum, whole colony and zooids; G. Lissoclinum cf fragile, whole colony; H. Polysyncraton sp, whole colony; I. Ascidia sydneiensis, with and without tunic; J. Ascidia cf. paratropa, with tunic; K. Ascidia sideralis, with and without tunic; L. Ascidia cf.munda, with and without tunic; M. Ascidia sp., three individuals with tunic; N. Ascidia ceratodes, with and without tunic; O. Pyura lignosa with and without tunic; P. Pyura bradleyi with tunic showing; Q. Pyura carmanae, with and without tunic; R. Pyura cf imesa with and without tunic; S. Microcosmus exasperatus with tunic; T. Herdmania cf. fimbriae with and without tunic; U. Polyandrocarpa sp. with and without tunic; V. Polyandrocarpa cf. zorritensis with tunic; W. Metandrocarpa taylori with tunic; X. Symplegma viridae with tunic; Y. Botrylloides nigrum with tunic. 15 16 Order Aplousobranchia Lahille, 1886 Family Diazonidae Seeliger, 1906 Genus Rhopalaea Philippi, 1843 Rhopalaea birkelandi Tokioka, 1971 Material examined: eight specimens (MZUCR-ASC-0235 - one ind.; Islita, 10.96466°N 85.69579°W; 8.00 m, attached to a rock; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0248 - one ind.; Isla Playa Rajada, 11.03356°N 85.75001°W; 11.00 m, between rocks; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0312 - one ind.; Mango, 10.95507°N 85.67398°W; 14.00 m, rocks fissures; 03/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0355 - one ind.; Palmares, 10.64501°N 85.68764°W; 8.00 m, on rock; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR- ASC-0396 - one ind.; Montosa, 10.57659°N 85.70300°W; 15.00 m, on rock; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0429 - one ind.; Jardín, 10.86252°N 85.91312°W; 4.60 m, on rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0471 - one ind.; Barco hundido, 10.9257°N 85.90321°W; 8.20 m, on sunken ship structure; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: The examined specimens add up the observations made by Tokioka (1972) and González-Sánchez et al., (2021a). The body is elongated, attached to the substratum by the posterior end of the body. The abdomen is short, and a thin peduncle is always present, but it usually stays in the substrate while removing it. The tunic is hard cartilaginous, with a smooth surface, free from any incrusting materials and brilliant purple in color. There are some individua.ls that show a light pink color, but do not differ morphologically (Fig. 2A). Substrate: Rocky bottom, porous rocks and cracks, and between algae. Reported distribution: Playas del Coco, Pacific, Costa Rica (Tokioka, 1972; Nova- Bustos et al., 2010; González-Sánchez et al., 2021), Isla Toboga and Isla Toboguilla, 17 Pacific, Panamá (Stoecker, 1980), Southern Gulf of Chiriquí, Pacific Panamá (Bullard et al., 2011). Family Polyclinidae Milne Edwards, 1841 Genus Polyclinum Savigny, 1816 Polyclinum cf. constellatum Savigny, 1816 Material examined: four specimens (MZUCR-ASC-0207, 208, 209, 213 - four different colonies; Bahía Thomas, 10.92949°N 85.71545°W; 6.00 m, floating net and pvc structure; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G. Alvarado, J.). The colony is grayish or light brown in color, roughly oval and attached to the substratum by a part of the lower side of the colony. The surface is smooth and free from incrustations. The zooids are visible beige and are arranged in several or many distinct systems. The small round-oval common cloacal orifices are distributed over the surface of the colony and have an irregular arrangement, at distances of a centimeter apart (Fig. 2B). Substrate: Rocky and sandy bottom. Distribution: Southern Gulf of California, México (Tovar-Hernández et al., 2010), Keehi Lagoon, Honolulu, Hawaii (Tovar-Hernández et al., 2010), Atlantic and Pacific sides of the Panamá Canal (Carman et al., 2011), Galápagos Islands (Lambert, 2019). Genus Aplidium Savigny, 1816 Aplidium cf. glabrum (Verrill, 1871) Material examined: three specimens (MZUCR-ASC-0168, 173, 195 - three different colonies; Pochote, 10.73134°N 85.79994°W; 6.40 m, on rocks with sand and algae; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J.). Observations: Match mostly the original described species Amouroucium glabrum (Verill1, 871), now Aplidium glabrum. The colony forms a thick, gelatinous, translucent and lobed tunic where the yellow-red zooids are conspicuous. The mature zooids have a 18 short stomach, with numerous folds and the intestine is larger in size usually containing seven to ten large, blackish focal pellets. Most of the zooid had ten stigmata rows (Fig. 2E). Substrate: Rocky and sandy bottom, shells, and walls. Distribution: Gulf of Maine, USA(Durante & Sebens, 1994), Kurile Islands, Rusia (Sanamyan, 2000), The Netherlands (Gittenberger, 2007). Family Polycitoridae Michaelsen, 1904 Genus Cystodytes Drasche, 1884 Cystodytes cf. dellechiajei (Della Valle, 1877) Material examined: four specimens (MZUCR-ASC-0183 - one colony; Pochote, 10.73134°N 85.79994°W; 5.00 m, on rock surface; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0236 - one colony; Islita, 10.96466°N 85.69579°W; 8.00 m, attached to rock; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0244, 257 - two colonies; La mesa, 11.02696°N 85.76276°W ; 6.00 m, on rock wall; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: Colonies follow the observations made by Van Name (1945). It is a flat incrusting colony with a smooth surface and slightly raised position of the zooids and not exceeding 5 mm thickness. All the individuals displayed a white-grayish color with a translucent tunic and making the calcareous disk-shaped spicules visible. When sliced open it is possible to observe that the spicules are often completely enclosing the bodies of the zooids except the thoracic part and in many cases, there are often scatter deposits of spicules in the tunic (Fig. 2D). Substrate: Rocky bottom, under rocks and shells. Distribution: Mediterranean sea (López-Legentil et al., 2005; Martínez-García et al., 2007), Rio de Janeiro, Brazil (R. M. da Rocha & Costa, 2005), Southeastern Australia (Łukowiak, 2012), Galápagos Islands (Lambert, 2019). 19 Family Didemnidae Giard, 1872 Genus Didemnum Savigny, 1816 Didemnum cf. perlucidum Monniot F., 1983 Material examined: four specimens (MZUCR-ASC-0247, 251 – two colonies.; Isla Playa Rajada, 11.03356°N 85.75001°W; 11.00 m, between rocks; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0381 - one colony.; Meros, 10.60914°N 85.68099°W; 7.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0404 – one colony; Pochote, 10.73134°N 85.79994°W; 11.20 m, under rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.) Observations: Individuals with small zooids of two body parts, between 4-5 stigmata rows and a large atrial aperture. Species confirmed by genetics (Cordon-Krumme et al. In prep.), difficulties encountered during dissection (Fig. 2C). Substrate: Rocky bottom and walls. Distribution: Matapalo reef, Pacific, Costa Rica (Roth et al., 2017a), Pacific coast of Panama (Carman et al., 2011), Galápagos Islands (Lambert, 2019). Genus Diplosoma Macdonald, 1859 Diplosoma listerianum (Milne Edwards, 1841) Material examined: four specimens (MZUCR-ASC-0210 – one colony; Bahía Thomas, 10.92949°N 85.71545°W; 6.00 m, floating net and pvc structure; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G. Alvarado, J., MZUCR-ASC-0350 - one colony.; Isla Pelonas, 10.57869°N 85.71055°W; 8.00 m, on rocks; 03/11/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0359 - one colony.; Palmares, 10.64501°N 85.68764°W; 8.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0383 - one colony.; Meros, 10.60914°N 85.68099°W; 7.00 m, on shell; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.,). Observations: It forms an extensive thin encrusting sheet with no spicules. Adjusting to the original (Milne Edwards, 1841) and subsequent (Ma et al., 2019) descriptions. 20 Colonies have a translucent tunic with scattered brown – greyish pigmented cells. Each zooid has a yellow pigmented body, four rows of stigmata and are grouped around a common exhalent opening. (Fig. 2F). Substrate: Rocky bottom and walls, dead corals, algae, and shells. Distribution: Tahiti Islands (Monniot 1987), Panama Canal (Carman et al., 2011), Galápagos Islands (Lambert, 2019), California and Australia (Susick et al., 2020). Genus: Lissoclinum Verrill, 1871 Lissoclinum cf. fragile (Van Name, 1902) Material examined: four specimens (MZUCR-ASC-0241- one colony.; La mesa, 11.02696°N 85.76276°W; 6.00 m, under rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0347 - one colony.; Isla Pelonas, 10.57869°N 85.71055°W; 8.00 m, on rock; 03/11/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0398 - one colony.; Montosa, 10.57659°N 85.70300°W; 15.00 m, on rock; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR- ASC-0461 – one colony.; Refugio, 10.9205°N 85.90231°W; 12.30 m, rock wall; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: We suggest the presence of this species, from the collected specimens they match the description provided by Tokioka (1972). We had difficulties in dissecting complete zooids due to the high level of contraction. A more thorough examination of these specimens must be attempted (Fig. 2G). Substrate: Rocky bottom and walls. Distribution: Pacific, Costa Rica (Tokioka, 1972), Panama Canal Pacific side (Carman et al., 2011). Genus Polysyncraton Nott, 1892 Material examined: two specimens (MZUCR-ASC-0249 - one colony; Isla Playa Rajada, 11.03356°N 85.75001°W; 11.00 m, over rock wall; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0255 - one colony; La cornuda, 21 11.01067°N 85.75162°W; 8.00 m, attached to a rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.) Observations: Encrusting bright orange colony up to 3 cm in diameter and up to 0.5 cm in height, zooids are not arranged in a system, and are up to 1 mm with atrial language divided. Zooids were difficult to dissect due to the small size of the colony and the high density of spicules. (Fig. 2H). Substrate: Rocky bottom and walls. Distribution: This genus is the second largest genus of didemnids, it has shown a wide distribution ranging from tropical to temperate waters (Oliveira et al., 2019). Order Phlebobranchia Lahille, 1886 Family Ascidiidae Herdman, 1882 Genus Ascidia Linnaeus, 1767 Ascidia sideralis Bonnet & Rocha, 2013 Material examined: six specimens (MZUCR-ASC-0221, 222 – two ind.; Muñecos, 10.58453°N 85.4254°W; 4.75 m, between rocks; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G. Alvarado, J., MZUCR-ASC-0238 - one ind.; Islita, 10.96466°N 85.69579°W; 8.00 m, attached to a rock; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0360 - one ind.; Palmares, 10.64501°N 85.68764°W; 8.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0403 – one ind.; Pochote, 10.73134°N 85.79994°W; 11.20 m, under rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0455 – one ind.; Refugio, 10.9205°N 85.90231°W; 12.30 m, rock fisures ; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: The collected individuals match the original description by Bonnet et al. (2013) and add up to the observations by González-Sánchez et al. (2021). The tunic goes from a white greyish to brown color, always accompanied by the presence of conspicuous white dots on the surface of the tunic. Individuals can be up to 8 cm with tunic and up to 5 cm without the tunic showing a blueish color, and each shows the musculature net on 22 the right side of the body with complete transverse fiber closer to the oral siphon (Fig. 2K). Substrate: Rocky bottom and walls, base of living corals and rock cracks. Distribution: Isla Canales de Tierra, Gulf of Chiriquí, Panama (Bullard et al., 2011; Bonnet et al., 2013), North Pacific of Costa Rica (González-Sánchez et al., 2021). Ascidia sydneiensis Stimpson, 1855 Material examined: five specimens (MZUCR-ASC-0214, 0218 – two ind.; Bahía Thomas, 10.92949°N 85.71545°W; 6.00 m, floating net and pvc structure; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G. Alvarado, J., MZUCR-ASC-0237 one ind.; Islita, 10.96466°N 85.69579°W; 8.00 m, attached to a rock; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0266 - one ind.; La cornuda, 11.01067°N 85.75162°W; 8.00 m, attached to a rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0298 - one ind.; Bajo Junquillal, 10.97687°N 85.69242°W; 4.00 m, under rock; 02/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: All de individuals collected follow the original description (Stimpson, 1855) and go along with the observations made by Bonnet & Rocha (2011) and González-Sánchez et al. (2021). Solitary individuals with a pale milky greyish tunic and distinctive muscles and fibers on the right side of the body (Fig. 2I). Substrate: Under rocks and between dead corals. Distribution: Guam (Lambert, 2002), Panamá Canal Pacific and Atlantic side (Carman et al., 2011), Galápagos Islands (Lambert, 2019), North Pacific of Costa Rica (González- Sánchez et al., 2021). Ascidia ceratodes (Huntsman, 1912) Material examined: one specimen (MZUCR-ASC-0095 - one ind.; Bajo Rojo, 10.5728°N 85.4401°W; 8.00 m, under rock; 01/viii/2018; col.; Ampié,G.; Flores, B.). 23 Observations: The description matches the one given by Tokioka (1972). The tunic is gelatinous, rather hard, faintly milky white, with a smooth surface and quite free from any foreign matters. The siphons are of a moderate length on the mantle body, the atrial siphon is slightly directed backwards. The body is whitish throughout, but faintly reddish orange in the siphonal areas (Fig. 2N). Substrate: Under rocks. Distribution: Playas del Coco, Pacific coast of Costa Rica (Tokioka, 1972; Nova-Bustos et al., 2010), Galápagos Islands (Lambert, 2019). Ascidia cf. munda Sluiter, 1898 Material examined: five specimens (MZUCR-ASC-0252, 0253 - two ind.; La cornuda, 11.01067°N 85.75162°W; 8.00 m, under rocks, together; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0280 - one ind.; Casa Verde Bajo Chaca, 10.98232°N 85.70425°W; 6.00 m, attached to rock side; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0330 - one ind.; Virador, 10.61076°N 85.70094°W; 12.00 m, attached under rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0376 - one ind.; Meros, 10.60914°N 85.68099°W; 7.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: Collected specimens match the original description (Kott, 1985). Long and narrow individuals, up to eight cm. The tunic is smooth, although there are some creases and ridges on the surface, and most of the time sand or muscles attached. It is firm and has a leathery light brown color with transparency. The body narrows to a long, terminal branchial siphon. The atrial siphon is usually from the middle or in the posterior half of the dorsal surface and directed posteriorly. The siphons have six longitudinal ridges corresponding to the six lobes. Long and crowded oral tentacles are present and the dorsal tubercle has a V shape. There is no musculature over the gut, which forms a deeply curved double loop, and the rectum is usually significantly expanded with fine mud. The anal border has four indentations and is not lobed (Fig. 2L). Substrate: Under rocks. 24 Distribution: Western Australia (Kott, 1985), Atlantic Panamá (Bonnet & Rocha, 2011). Ascidia cf. paratropa (Huntsman, 1912) Material examined: one specimen (MZUCR-ASC-0226 - one ind.; Islita, 10.96466°N 85.69579°W; 8.00 m, attached under rock; 15/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J.). Observations: The collected individual adds up to the description provided by Van Name (1945). Apart from being cylindrical and attached by the posterior end, the colorless tunic exhibited prominent and conspicuous tubercles of different sizes having a blue-purple tip (Fig. 2J). Substrate: Under rocks. Distribution: Western Australia (Kott, 1985). Order Stolidobranchia Lahille, 1886 Family Pyuridae Hartmeyer, 1908 Genus Pyura Molina, 1782 Pyura lignosa Michaelsen, 1908 Material examined: six specimens (MZUCR-ASC-0224 – one ind.; Muñecos, 10.58453°N 85.4254°W; 4.75 m, on top of rock; 15/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0259, 0261 - two ind.; La cornuda, 11.01067°N 85.75162°W; 8.00 m, attached to rock side; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0369 0355 - one ind.; Palmares, 10.64501°N 85.68764°W; 8.00 m, on rock; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0407 – one ind.; Pochote, 10.73134°N 85.79994°W; 11.20 m, under rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR- ASC-0435 - one ind.; Jardín, 10.86252°N 85.91312°W; 4.60 m, on rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: All specimens were firmly fixed to the substrate by a leathery tunic as thick as 1.5 cm and followed the original description from Michaelsen (1908) and they 25 add up to the observations made by Monniot (1994), Rocha & Counts (2019), González- Sánchez et al. (2021) (Fig. 2O). Substrate: Growing over rocks and walls, sometimes where there is abundant algae. Distribution: Panamá canal Pacific side (Carman et al., 2011), North Pacific of Costa Rica (González-Sánchez et al., 2021). Pyura stolonifera (Heller, 1878) Material examined: five specimens (MZUCR-ASC-0180 - one ind.; Pithaya, 10.97759°N 85.80171°W; 5.00 m, under rock; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.; Alvarado, J., MZUCR-ASC-0195, 0199 – two ind.; Pochote, 10.73134°N 85.79994°W; 11.20 m, between rocks; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0274 - one ind.; La mesa, 11.02696°N 85.76276°W ; 6.00 m, rocks with algae coverage; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0450 - one ind.; Mogotes, 10.95574°N 85.90321°W; 13.20 m, rocky bottom; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: The tunic is leathery opaque usually covered with sand, algae and smalls shells, concordant with the original description by Van Name (1931) (Fig. 2P). Substrate: Under and between rocks covered with algae. Distribution: Western Australia (Monteiro et al., 2002), Southern Cape, South Africa (Fielding et al., 1994). Pyura carmanae Rocha & Counts, 2019 Material examined: eight specimens (MZUCR-ASC-0090 - one ind.; Bajo la vita, 10.89056°N 85.91589°W ; 13.00 m, on rock wall; 17/vii/2019; col.; Alvarado, J.; Ampié, G.;Flores, B.; Quesada, F., MZUCR-ASC-0186, 0189 - two ind.; Pithaya, 10.97759°N 85.80171°W; 5.00 m, under rock; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.; Alvarado, J., MZUCR-ASC-0268 - one ind.; La cornuda, 11.01067°N 85.75162°W; 8.00 m, attached to a rock; 01/ix/2023; col.; Cordón, I; González, K; 26 Chacón, L; Ampié, G., MZUCR-ASC-0286 0280 - one ind.; Casa Verde Bajo Chaca, 10.98232°N 85.70425°W; 6.00 m, attached to rock side; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0335 - one ind.; Virador, 10.61076°N 85.70094°W; 12.00 m, attached under rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0356 - one ind.; Palmares, 10.64501°N 85.68764°W; 8.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0406 – one ind.; Pochote, 10.73134°N 85.79994°W; 11.20 m, under rock; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: The collected individuals followed the original description of (Rocha & Counts, 2019) as mentioned by (González-Sánchez et al., 2021). The living individuals show a yellow wrinkled leathery tunic and are usually 2-6 cm. Both siphons are apical and close together; these can show a red, orange strong coloration. Some individuals show larger siphons than the original description, suggesting a possible variation (Fig. 2Q). Substrate: Mostly under rocks and rock cracks. Distribution: Costa Rica (Tokioka, 1972), Gulf of Chiriquí, Isla Canales de Tierra, Panama (Bullard et al., 2011; R. M. Rocha & Counts, 2019). Pyura cf. imesa Rocha & Counts, 2019 Material examined: eight specimens (MZUCR-ASC-0109 – one ind.; Los Cabros, 10.94497°N 85.80185°W; 6.00 m, under rock; 16/ix/2019; col.; Alvarado, J.; Ampié, G.;Flores, B.; Quesada, F., MZUCR-ASC-0178 - one colony; Pochote, 10.73134°N 85.79994°W; 5.00 m, on rock surface; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0190 - one ind.; Pithaya, 10.97759°N 85.80171°W; 5.00 m, under rock; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.; Alvarado, J., MZUCR-ASC-0342, MZUCR-ASC-0451 - one ind.; Mogotes, 10.95574°N 85.90321°W; 13.20 m, rocky bottom; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0476 - one ind.; Barco hundido, 10.9257°N 85.90321°W; 8.20 m, on sunken ship structure; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). 27 Observations: The examined specimens match most of the description made by Rocha & Counts (2019). The body wall is vivid orange with a strong musculature around the siphons and longitudinal muscles radiate from the siphons forming a continuous sheet. There is a large velum in the oral siphon, and it has between 25-28 oral tentacles of two orders. The dorsal tubercule is a distinctive thin V-shape, and the dorsal lamina is divided into languets. The pharynx presents six folds per side and with 9 or 10 stigmata per mesh between and on folds. The vessel formula is from right to left (total 240): E 2 (12) 8 (15) 7 (15) 5 (14) 6 (13) 6 (12) 6 DL 4 (16) 5 (15) 5 (17) 6 (17) 5 (15) 6 (10) 3 E The digestive gland is relatively small and brown, exhibiting tubular projections. The morphology of the gonads is the same as in the image shown by Rocha & Counts (2019), but the individuals show more abundance of genital capsules up to 35 per side (Fig 2R). Substrate: Under rocks and rock cracks. Usually seen with P. carmanae Distribution: North Pacific of Costa Rica (Tokioka, 1972; González-Sánchez et al., 2021), Isla Canales de Tierra, Panama (Rocha & Counts, 2019). Genus Microcosmus Heller, 1877 Microcosmus exasperatus Heller, 1878 Material examined: four speciemns (MZUCR-ASC-0077, 0105 – two ind.; Bahía Thomas, 10.5547°N 85.4254°W; 6.00 m; 02/viii/2018; col.; Ampié, G; Flores, B., MZUCR-ASC-0390 - one ind.; Meros, 10.60914°N 85.68099°W; 7.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0477 - one ind.; Barco hundido, 10.9257°N 85.90321°W; 8.20 m, on sunken ship structure; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: Individuals do not exceed 5 cm, and are almost always covered with algae or sand; it follows the original description and the observations made recently by Van Name (1945) and González-Sánchez et al. (2021a). We confirm the identification we observed the presence of velum and the spines in the oral siphon (Fig. 2S). 28 Substrate: Between and under rocks. Distribution: Galápagos Islands (Lambert, 2019). Genus Herdmania Lahille, 1888 Herdmania cf. fimbriae Kott, 2002 Material examined: one specimen (MZUCR-ASC-0481 - one ind.; Matapalo, 10.5347°N 85.7614°W; 9.00 m, between dead corals and algae (Hypnea sp.); 04/vii/2024; col.; Cordón, I; González, K; Quesada, F.). Observations: Collected individuals add up to the original description by Kott (2002). Organisms grow up to 4 cm and have a pink, translucent, leathery and opaque tunic. Both siphons originate close to one another on the upper surface. Spines are present, longitudinally arranged and parallel in siphons, but in the body wall they are randomly oriented. The dorsal tubercule has a U-shaped slit with horns turned in. The branchial sac has 8-9 broad, longitudinal folds on each side (Fig. 2T). Substrate: Between dead corals. Distribution: Australia (Kott, 2002) Family Styelidae Herdman, 1881 Genus Polyandrocarpa Michaelsen, 1904 Polyandrocarpa cf. zorritensis (Van Name, 1931) Material examined: three specimens (MZUCR-ASC-0385 - one ind.; Meros, 10.60914°N 85.68099°W; 7.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0428 - one ind.; Isla Cocinera, 10.85825°N 85.91312°W; 4.10 m, on rock upper surface; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0469 - one ind.; Barco hundido, 10.9257°N 85.90321°W; 8.20 m, on sunken ship structure; 25/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: It is an encrusting and flat colony and follows the original description made by Van Name (1931). The tunic is leathery, the zooids are vertical and both siphons are opening to the surface (Fig. 2V). 29 Substrate: Rocky bottom. Distribution: Gulf of Mexico, Florida, Bahia to Santa Catrina, Brazil (Rocha et al., 2012). Genus Metandrocarpa Michaelsen, 1904 Metandrocarpa taylori Huntsman, 1912 Material examined: six specimens (MZUCR-ASC-0181 - one ind.; Pithaya, 10.97759°N 85.80171°W; 5.00 m, under rock; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.; Alvarado, J., MZUCR-ASC-0239 - one ind.; La mesa, 11.02696°N 85.76276°W ; 6.00 m, rocks with algae coverage; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0293 - one ind.; Bajo Junquillal, 10.97687°N 85.69242°W; 4.00 m, under rock; 02/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0299 - one ind.; Bajo Viejón, 10.95507°N85.72843°W; 17.00 m, between rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0394 - one ind.; Montosa, 10.57659°N 85.70300°W; 15.00 m, on rock; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0457 – one ind.; Refugio, 10.9205°N 85.90231°W; 12.30 m, rock fisures ; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: Follows Huntsman (1912) original description and the annotations made by Van Name (1945). It is a colonial species, thin, encrusting and the individuals are connected by a stolon not by the tunic, and have a hemispherical shape, dark red or bright cherry-red color. The surface of the tunic is smooth or slightly wrinkled. The zooids do not have folds in the branchial basket (Fig 2W). Substrate: Rocky bottom, walls and cracks, dead coral. Distribution: Metandrocarpa taylori for China, Brithish Columbia and M. michaelseni from California (Van Name, 1945). Genus Symplegma Herdman, 1886 Symplegma brakenhielmi (Michaelsen, 1904) 30 Material examined: six specimens (MZUCR-ASC-0171 - one colony; Pochote, 10.73134°N 85.79994°W; 5.00 m, on rock surface; 14/iv/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G; Alvarado, J., MZUCR-ASC-0242 - one ind.; La mesa, 11.02696°N 85.76276°W ; 6.00 m, under rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0291 - one ind.; Bajo Junquillal, 10.97687°N 85.69242°W; 4.00 m, under rock; 02/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0338 - one ind.; Virador, 10.61076°N 85.70094°W; 12.00 m, attached under rock; 01/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0373 - one ind.; Meros, 10.60914°N 85.68099°W; 7.00 m, on rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0456 – one ind.; Refugio, 10.9205°N 85.90231°W; 12.30 m, between rocks; 26/vii/2024; col.; Cordón, I; González, K; Chacón, L; Ampié, G.). Observations: The original description matches our individuals (Michaelsen, 1904). It is an encrusting colony with oval shaped zooids with dark olive green and grey, having a conspicuous siphon and a variety of sizes, shapes and colors. (Fig. 2X). Substrate: Growing under and over rocks, covering walls and going between cracks. Distribution: Bermuda (Van Name, 1931), Caribbean Sea (Palomino-Alvarez et al., 2022). Genus Botrylloides Milne Edwards, 1841 Botrylloides cf. niger Herdman, 1886 Material examined: five specimens (MZUCR-ASC-0140, 0143 – two ind.; Muñecos, 10.58453°N 85.4254°W; 4.75 m, on top of rock; 15/ix/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G., MZUCR-ASC-0160, 0162 – two ind.; Isla David, 10.5727°N 85.4313°W; 4.60 m, on top of rock; 23/vi/2021; col.; Chacón, L; Flores, B., MZUCR- ASC-0308 - one ind.; Bajo Viejón, 10.95507°N85.72843°W; 17.00 m, between rocks; 04/xi/2023; col.; Cordón, I; González, K; Chacón, L; Ampié, G.,). 31 Observations: The original description matches our individuals Herdman (1886) (Fig. 2Y). Zooids are completely embedded in the tunic and are organized in a meandering system around the common cloaca. Substrate: Rocky bottom, covering rocks and over algae. Distribution: Galápagos Islands (Lambert, 2019), Panamá canal Pacific side (Carman et al., 2011). Discussion Species identification and site diversity Of the 34 species identified, 19 are colonial and 14 are solitary. Most of the colonial species belong to the order Aplousobranchia, except Rhopalaea birkelandi being solitary within the order. In the Phlebobranchia order all the species were solitary and, in the order Stolidobranchia all species from the Pyuridae family were solitary and colonial species were found in the Styelidae family. The inclusion of eight new records for the northern coast of Costa Rica: Aplidium cf. glabrum, Ascidia cf. munda, Ascidia cf. paratropa, Hermania cf. fimbriae, Pyura cf. imesa, Botrylloides cf. nigrum, Metandrocarpa taylori, Polyandrocarpa cf. zorritensis and Symplegma brakenhielmi represents a significant contribution to the region's documented ascidian biodiversity. These findings indicate that the ascidian community in this area is more complex and less explored than previously understood, providing essential baseline data for future studies. During our surveys we observed high abundance of R. birkelandi and P. carmanae in the same sampling site, it has been determined that Pyura sp. is found in the same habitats as R. birkelandi (Stoecker, 1980). In other hand we did not observe crustaceans inside the R. birkelandi individuals but we did observe few shrimps and crabs living inside the P. lignosa individuals (Fig S3). Fujino (1972). found that they can have associated commensals, in this case a shrimp species of the genus Pontia. There has been determined that the pinnotherid crab Tunicotheres moseri was frequently found as a commensal in the atrial cavity of Phallusia nigra, from the Caribbean (Goodbody, 2003). 32 Polyandrocarpa anguinea, a species previously recorded in surveys by González-Sánchez et al. (2021), was notably absent in our 2023 and 2024 surveys, despite revisiting the same sites and conducting additional sampling points. In contrast, other species such as Polyandrocarpa cf. zorritensis and unidentified specimens were documented, providing valuable new records for the northern Pacific of Costa Rica. These findings underscore gaps in current knowledge and emphasize the importance of updated taxonomic studies to improve understanding of the region’s biodiversity. Species Status Assessment The status evaluation revealed that eight species are considered introduced, six cryptogenic, eight native, and ten undetermined, reflecting the limited knowledge we have on ascidian diversity along the north Pacific coast of Costa Rica. The presence of introduced species, such as Polyclinum constellatum and Didemnum cf. perlucidum, raises concerns about their impact on native biodiversity and the potential for ecosystem disturbance (Smale & Childs, 2012). These invasive species could outcompete native ascidians for resources, alter habitats, and introduce new pathogens. Polyclinum constellatum has been recorded together with a parasitic cyclopoid copepod Haplostomides hawaiiensis Ooishi,1994 (Tovar-Hernández et al., 2010). In an experiment conducted in Costa Rica, D. perlucidum was reported and demonstrated invasive behavior, consistently colonizing settlement tiles across all treatments, and on coral reefs environments (Roth et al., 2017a), a trait typical of many invasive ascidian species (Kremer et al., 2010). Didemnum cf. perlucidum is now widespread in tropical waters, presumed to be of Atlantic Ocean origin (Kremer et al., 2010). The classification of this species is challenging due to the lack of historical data and the difficulties associated with morphological identification. It was originally described as native to the Caribbean, which is why we believe it was introduced to Costa Rica. Its distribution may be expanding in response to factors such as increased shipping and aquaculture, rising temperatures, and eutrophication (Lambert, 2007). Diplosoma listerianum has been reported in proximity to the Panama Canal (Carman et al., 2011), the Galápagos Islands (Lambert, 2019) and California (Susick et al., 2020), 33 which further increases the likelihood of its occurrence in the regions. Recently it has been reported to Brier Island, Nova Scotia, Canada (Ma et al., 2018). The presented description matches our observation on the colonies and individuals. Cryptogenic species are those whose origins remain uncertain, they cannot be definitively classified as alien or native (Carlton, 1996; Minchin, 2009). In our study, six species were classified as cryptogenic, characterized by ambiguous taxonomy and identification challenges. For example, Pyura bradleyi is now recognized as Pyura stolonifera, a species with taxonomic uncertainties due to numerous synonymized names. Its reported native range is limited to the southern part of Africa (Fielding et al., 1994). However, our observations are most consistent with Van Name's (1931) description, highlighting the need for reevaluation. In addition, P. stolonifera is predominantly found on landmasses that were once part of the Gondwanan supercontinent, which fragmented during the Mesozoic. This biogeographic context suggests that populations in Chile and other parts of South America were likely introduced, as these regions were never historically connected to its native range (Rius & Teske, 2011). P. anguinea, previously documented by González-Sánchez et al. (2021), was not observed during our recent sampling events. This species has been recorded on the Atlantic side of the Panama Canal (Carman et al., 2011) and at a few locations along the Caribbean, making its origin difficult to determine and leading to its classification as a cryptogenic species. In contrast, during the most recent sampling at the Barco Hundido (BH) site, numerous specimens of P. cf. zorritensis were observed. This species has been reported in the Pacific region, including the Panama Canal and the Galapagos Islands, supporting its classification as an introduced species (Carman et al., 2011; Lambert, 2019). Aplidium cf. glabrum has been reported primarily in the Caribbean region (Durante & Sebens, 1994; Sanamyan, 2000; Gittenberger, 2007), raising the possibility that it may have been introduced to other areas. However, in the absence of conclusive evidence to confirm its origin, we classify this species as cryptogenic. Native species, on the other hand, play a critical role in maintaining the balance and resilience of marine ecosystems. Identifying native species and understanding their 34 ecological roles can help establish baselines for assessing the impacts of introduced and cryptogenic species (Wares et al., 2005). The eight native species recorded in this study underscore the richness of natural ascidian diversity in the region and suggest a potentially greater diversity that remains undocumented. The protection of native species is essential for the preservation of ecosystem functionality, as they are adapted to local conditions and have complex relationships with other marine organisms. Finally, the ten species with undetermined status highlight the need for continued research and monitoring to accurately classify these species and assess their potential impacts on local biodiversity. As more data becomes available, it will be possible to refine management strategies and ensure the protection of native species and the overall health of the marine environment. Understanding the interactions between these species and their environment is crucial for developing effective management strategies to protect native biodiversity. Given the presence of both introduced and cryptogenic species, it is essential to explore strategies for managing these populations, such as implementing monitoring programs, enforcing biosecurity measures, and undertaking habitat restoration efforts. Conducting a comprehensive study to understand the ecological dynamics among introduced, cryptogenic, and native species will be vital for preserving the health and diversity of the local marine environment. Morphological variations among species Identification of ascidians based on morphological characters is challenging due to the high variability within and between species. This process requires considerable taxonomic expertise, precise dissection skills, and access to detailed species descriptions, which are often scarce for certain regions (Stefaniak, 2009). Morphological characters such as coloration, siphon position, spicule shape, and zooid structure can vary significantly not only between closely related species, but also within the same species, leading to ambiguity in classification. Following this we address these challenges by discussing specific examples of species with complex or ambiguous taxonomy. Cases such as Pyura cf. imesa and Cystodytes illustrate how subtle morphological differences and intraspecific variability complicate 35 identification. Similarly, discrepancies in species descriptions, such as those for Aplidium cf. glabrum and Ascidia cf. munda, underscore the importance of revisiting original descriptions and integrating genetic tools for accurate classification. This discussion underscores the need for a more thorough examination of ascidian taxonomy and highlights the limitations of relying solely on morphological characters, especially in regions with limited taxonomic resources. For the Pacific region it has been reported R. birkelandi with a bright blue color and no pink (Bullard et al., 2011), suggesting other color morphotype. Rhopalaea presents a very thin peduncle, separating the zooids into two distinct parts, and it has been determined that this part of the body allows some Aplosuobranchia species to lose their thorax when environmental conditions are harsh and regenerate it when conditions improve (Shenkar et al., 2016). A thorough and detailed review of morphological characters, including coloration, peduncle structure, and taxonomy, is essential to determine whether the observed differences represent a color variation within R. birkelandi or indicate the presence of a distinct morphotype or species. González-Sánchez et al. (2021) noted that three specimens of P. carmanae differ in the position of the siphons. Upon revising these specimens and incorporating our own sampling data, we propose that one of these specimens corresponds to Pyura cf. imesa (Rocha & Counts, 2019). While our individuals exhibit a greater number of oral tentacles and gonadal lobes, we also suggest that the specimens described by Tokioka (1972) as P. lignosa likely belong to this species (P. cf. imesa). Regarding the remaining morphotypes, we propose they represent a possible variation of P. carmanae, with the main distinctions being the size and position of the siphons. Metandrocarpa taylori could be confused with M. dura, but from the beginning the described characters, such as size, the presence of dome-shaped elevations, the leathery tunic and the number of oral tentacles doesn’t match. In the book from North and South American ascidians, one of the annotations made was that M. taylori could be also confused with M. michaelseni, possibly because these are not distinct, so in case of doubt M. taylori should be used (Van Name, 1945). 36 Despite the highly variability in spicule shapes in ascidians, the individuals Cystodytes showed disk-shaped spicules very similar to the ones showed in a study from Australia (Łukowiak, 2012). Although the first record for C. dellechiajei was on the Brazilian coast, it was soon considered a widespread and even a cosmopolitan species with high intraspecific variability (Kott, 1990; López-Legentil & Turon, 2005). In the last years many morphotypes have been identified, most of which vary in colors and spicule composition, making difficult to determine species just morphologically (López-Legentil & Turon, 2005; Rocha & Kremer, 2005). Aplidium cf. glabrum has been reported mainly for the Caribbean (Durante & Sebens, 1994; Sanamyan, 2000; Gittenberger, 2007). For Playas del Coco on the Pacific coast of Costa Rica Tokioka (1972) reported Amaroucium constellatum, now Aplidium constellatum, but the description of the collected specimens does not match because this species has more than ten stigmata rows. The report mentions the possibility of being similar to A. californicum (Ritter & Forsyth, 1917), but this species is much smaller and the size and position from the languet are completely different. We do not discard the possibility; while making the identification you can find some difficulties while extracting and dissecting the zooids and mostly when there is strong contraction. Since the original description by Kott (1985) holds more weight, the discrepancies between Ascidia cf. munda described by Bonnet & Rocha (2011) and our specimen could be attributed to geographic differences. The specimen described here is from the Atlantic, whereas Ascidia munda is known from the Pacific Ocean, following Kott’s (1985) original description. Herdmania cf. fimbriae follows the original description (Kott 2002) and the remarks on the differences with other species made are accurate, plus H. momus and H. pallida have mostly a Caribbean and Atlantic distribution. The identification of ascidian species based solely on morphological characters remains challenging due to the inherent variability of these traits and the limited taxonomic tools available. By relying on original descriptions and regional resources from North and South America, we have established a reliable basis for our assessments. However, further 37 research, molecular integration, and comprehensive revisions are needed to refine classifications and enhance our understanding of ascidian biodiversity in the north Pacific. Conclusion This study provides a comprehensive overview of the ascidian diversity along the 32 sampling sites in the northern Pacific coast of Costa Rica, specifically within the Guanacaste Conservation Area (ACG). A total of 456 ascidian specimens were collected between 2019 and 2024, representing 33 species across 16 genera, seven families, and three orders within the class Ascidiacea. We also include eight new records: Aplidium cf. glabrum, Ascidia cf. munda, Ascidia cf. paratropa, Hermania cf. fimbriae, Pyura cf. imesa, Botrylloides cf. nigrum, Metandrocarpa taylori, Polyandrocarpa cf. zorritensis and Symplegma brakenhielmi, which signifies a considerable contribution to the region's documented ascidian biodiversity. Furthermore, the presence of introduced and cryptogenic species, alongside native ones, underscores the need for ongoing monitoring to understand species distribution and dynamics. 38 Capítulo 2. Want to know about a relative of ours? Phylogenetic relationships among solitary and colonial ascidians (subphylum Tunicata; class Ascidiacea) from the northern Pacific of Costa Rica María Isabel Cordón-Krumme1,2 https://orcid.org/0000-0001-5974-5865 Melissa Mardones-Hidalgo 2 https://orcid.org/0000-0002-4402-7817 Laura Brenes-Guillén 2,4 https://orcid.org/0000-0002-7185-4084 Kaylen González-Sánchez1,4 http://orcid.org/0000-0002-7208-9302 Juan-José Alvarado-Barrientos1,2,3 http://orcid.org/0000-0002-2620-9115 1 Centro de Investigación en Ciencias del Mar y Limología (CIMAR), Universidad de Costa Rica, 11501- 2060, San José, Costa Rica 2 Escuela de Biología, Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 3 Museo de Zoología, Centro de Investigación en Biodiversidad y Ecología Tropical (CIBET), Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 4 Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, 11501- 2060, San José, Costa Rica Abstract | Ascidiacea, a class within the subphylum Urochordata, is the sister group to other chordates, despite having a distinct morphology. However, taxonomic identification of ascidians is complex due to morphological similarities between species. In this study, we investigate the phylogenetic relationships of ascidian species from the northern Pacific Ocean of Costa Rica, using both morphological and molecular data. Field sampling at 31 sites identified 34 species belonging to three orders. DNA was extracted and two genetic markers, mtCOI and 18s rRNA, were amplified and sequenced. Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI), showing strong support for the major taxonomic orders and demonstrating the reliability of mtCOI in resolving phylogenetic relationships within Ascidiacea. Our results contribute to the understanding of ascidian diversity in Costa Rica, highlight their evolutionary relationships, and emphasize the importance of integrative approaches in resolving tunicate taxonomy Key words: Ascidiacea, Pacific Ocean, 18s RNA, mtCOI, Phylogeny https://orcid.org/0000-0001-5974-5865 https://orcid.org/0000-0002-4402-7817 https://orcid.org/0000-0002-7185-4084 http://orcid.org/0000-0002-7208-9302 http://orcid.org/0000-0002-2620-9115 39 Introduction It has been known that tunicates belong to chordates, being the closest relative to vertebrates (Bourlat et al., 2006; Tsagkogeorga et al., 2009). The class Ascidiacea belongs to the subphylum Urochordata and is characterized for having basic characteristics of a chordate (postanal tail, dorsal nerve cord, notochord, gill slits, and endostyle) (Monniot et al., 1991; Satoh et al., 2014). However, they lack a backbone and instead possess a notochord only during larval stage, establishing that they possess a prototypical chordate body plan (Corbo et al., 2001; Bastida-Zavala et al., 2014). They are a group of exclusively marine benthic, sessile, hermaphrodite group of animals with irregular spherical body morphology (Monniot et al., 1991). The class Ascidiacea (from the Greek "small sac") is the most diverse group of the subphylum Tunicata with approximately 3,000 species divided into three orders based primarily on the internal structure of the branchial sac: Aplousobranchia (simple), Stolidobranchia (folded) and Phlebobranchia (vascular). Currently, three types of life organization have been described: solitary, colonial and mixed (Lahille, 1886; Shenkar & Swalla, 2011).The basic body plan of this class is composed of three layers arranged one inside the other: the outer layer known as the tunic is composed of cellulose, the intermediate layer called the mantle, and the inner layer comprising the gill sac (Monniot et al., 1991; Corbo et al., 2001). These layers have two openings, the oral siphon, through which food enters, and the anal siphon, through which they expel digestion residues and gametes (Monniot & Monniot, 1978). Ascidians feed by active filtration, mainly on plankton and occupy a central role in the ecology and dynamic of marine benthic communities (Lambert, 2005; Petersen, 2007). The interest in ascidian studies has grown, especially in evolutionary processes, given their close relationship with vertebrates (Cleto et al., 2003; Lemaire et al., 2008). The fact that draws the scientist attention it’s the association with bacteria allowing the ascidians to generate secondary metabolites with antibacterial and anti-inflammatory activity, with pharmaceutical and biotechnological applications (Blunt et al., 2016; Carroll et al., 2020). There has been some success with two ascidians natural products marketed as therapeutic 40 drugs for cancer, one is known as Yondelis® from Ecteinascidia turbinata and the second one is Aplidin® first isolated from Aplidium albicans (Palanisamy et al., 2017; Watters, 2018). In addition numerous ascidians have become successfully introduced around the globe via anthropogenic vectors, giving them significant importance (Zhan et al., 2015). In recent years has been established that there are many invasive ascidian species, with a strong ability to spread rapidly, caused by the tolerance to a wide range of temperatures and salinities (Carver et al., 2006; Sheets et al., 2016; Nydam et al., 2021). However, their frequent invasions in coastal habitats are leading to economic and ecological damages, causing declines in native species richness, altering benthic community structure and disrupting links between benthic and pelagic communities (Locke et al., 2009; Aldred & Clare, 2014; Palanisamy et al., 2017; Giachetti et al., 2020; Platin & Shenkar, 2023). Despite the high diversity and the global mobility of several species, the systematics and taxonomy of ascidians are not well resolved (Rocha et al., 2019; Nydam et al., 2021). It is difficult to identify taxonomically ascidians because of the similarity in the characteristics, making it very hard to tell the differences among species. Besides the morphological-based species identification is still a challenge despite all the considerable efforts (Van Name, 1945; Rocha et al., 2019). Recently, studies that combine molecular and morphological analyses have revealed new species and validated the taxonomy of already described species (Zeng et al., 2006; Bonnet & Rocha, 2011; Rocha et al., 2019; Salonna et al., 2021). To date are several studies about species identification and the elucidation of phylogenetic relationships among species within the class Ascidiacea and among classes (Kurabayashi et al., 2003; Shenkar & Swalla, 2011; Akram et al., 2018; Giribet, 2018; Nydam et al., 2023). In the last years, scientists have increased the use of molecular phylogenetic analyses to understand tunicate relationships. Building upon Wada (1998) pioneer study, researchers have predominantly relied on the 18S rRNA gene as a key marker for reconstructing tunicate relationships across different taxonomic scales (Swalla et al., 2000; Stach & Turbeville, 2002; Tsagkogeorga et al., 2009; Giribet, 2018; Nydam et al., 2021, 2023). These molecular phylogenies consistently challenge traditional classification, particularly at higher taxonomic levels. Nevertheless, it has been determined that within the Tunicata, 41 the conserved 1kb fragment of the 18s RNA resolves the relationship between ascidian families much better than mitochondrial genetic material (Wada, 1998; Stach & Turbeville, 2002; Zeng et al., 2006; Ananthan & Murugan, 2018). The mitochondrial cytochrome c oxidase subuinit I (Cox1), has been the molecule of choice in studies of population genetics and to address cryptic speciation and invasions, due to its high variability (Turon & López-Legentil, 2004; Ananthan & Murugan, 2018). However, the mtCOI gene may provide useful information at higher taxonomic levels (Turon & López- Legentil, 2004; Ananthan & Murugan, 2018). For Costa Rica, studies related to the morphological and molecular identification of ascidian species are scarce (Tokioka, 1972; Nova-Bustos et al., 2010; Nova Bustos et al., 2016; Gonzalez-Pestana et al., 2017; Roth et al., 2017b; González-Sánchez et al., 2021). In this study we aim to elucidate the phylogenetic relationships among the species of the Class Ascidiacea present in Northern Pacific of Costa Rica, providing insights through morphological and molecular identification. Materials and methods The sampling took place during four field expeditions of 4 days during April, August, November 2023 and July 2024. The 31 sampling sites were located at Costa Rica´s North Pacific region and a total of 34 species were identified, corresponding to 15 genera, seven families (Ascidiidae, Diazonidae, Didemnidae, Polycitoridae, Polyclinidae, Pyuridae and Styelidae), and three orders within class Ascidiacea. Genetic identification DNA was extracted from all specimens using the GeneJET Genomic DNA Purification Kit (Thermo Scientific), following the commercial protocol with a few modifications. These included a lysis time of 48 hours and the use of 25 µL of Proteinase K and RNase A solution. For solitary ascidians, DNA was extracted from a tissue sample taken from the oral siphon, while for colonial specimens, DNA was extracted from dissected zooids. The mtCOI fragment was amplified using the standard Folmer´s primers LCO1490 and HCO2198 (Folmer et al., 1994). PCR amplifications were performed using a Veriti 96 42 Well Thermal Cycler from Applied Biosystems) in a 25 uL reaction volume containing: 2.5 µL of 1x reaction buffer with 1 mM final concentration of 0.10 µL DreamTaq, 2.5 µL of DNTPs, 1 µL of each of the two primers, 1 µL BSA, and 15.9 ultra-pure water. Amplification conditions were three minutes at 95°C for denaturation followed by 35 cycles with annealing for 45 seconds at 94°C, 45 seconds at 45°C, and extension for two minutes at 72°C, followed by a final elongation step of ten minutes at 72°C and hold at 4 °C. The 18Sr RNA fragment was amplified using the 18s1 y 18s4 primers (Tsagkogeorga et al., 2009). Amplifications were carried out in a 25 µL reaction volume containing: 2.5 µL of 1x reaction buffer with 1 mM final concentration of 0.10 µL DreamTaq, 2.5 µL of DNTPs, 1 µL of each of the two primers, 1 µL BSA, and 15.9 ultra-pure water. The amplification protocol used consisted of 1 min at 94°C for initial denaturation followed by 30 cycles of 10 s at 98°C, 50s at 50°C, 2 min at 72°C, and a final elongation step of 10 min at 72°C. 18 s PCR products were visualized on a 1% TAE agarose gel stained with GelRed (Nucleic Acid Gel Stain) under UV illumination (Ruiz et al., 2020) and were outsourced for sequencing to Macrogen Corporation (Korea) on an ABI3730XL automatic DNA sequencer. The mtCOI sequences were obtained through The Barcode of Life Data System (BOLD) (http://www.barcodinglife.org), an integrated bioinformatics platform that supports all phases of the analytical pathway from specimen collection to tightly validated barcode library (Ratnasingham & Hebert, 2007). Phylogenetic analyses The obtained raw sequences were uploaded to Benchling software, for each forward and reverse sequence the electropherogram was viewed, then edited and aligned to create the consensus sequence. After obtaining all the consensus sequences for both genes, they were compared to the nucleotide non-redundant database using BlastN (https://blast.ncbi.nlm.nih.gov/Blast.cgi), extracting the genetic identification, the accession number, percentage of identity, e value and the query coverage for each sample (Table S4 and S5). http://www.barcodinglife.org/ https://blast.ncbi.nlm.nih.gov/Blast.cgi 43 The Coi dataset contained the 156 newly generated sequences, 14 sequences obtained from Gene Bank and 1 Outgroup (Branchiostoma floridae). The 18s rRNA dataset contained the 68 new generated sequences and 1 Outgruop (Branchiostoma floridae). All the obtained sequences were aligned using MAFFT v7.023b (Katoh & Standley, 2013) with E-INS-I iterative refinement methods, recommended for datasets with less than 200 sequences with multiple conserved domains and long gaps. Alignments were inspected visually in AliView (Larsson, 2014) to ensure that the settings used in the previous procedure were appropriate, avoiding misaligned regions. The sequences obtained from GenBank are shown in Table 2. and sequences of the cephalochordate Branchiostoma floridae mtCOI (NC_000834.1/ AF098298.1) and 18s rRNA(M97571.1) were used as Outgroup, just as other studies (Stach & Turbeville, 2002; Moreno & Rocha, 2008; Hasegawa et al., 2024) Table 2. Species whose mtCOI sequences were used the phylogenetic analysis of this study. Species Order Family mtCOI Acc. No. Amaroucium stellatum Aplousobranchia; Synoicidae AY116595.1 Cystodytes dellechiajei Aplousobranchia Polycitoridae AY523068.1 Ascidia sydneiensis Phlebobranchia Ascidiidae MT637975.1 Symplegma brakenhielmi Stolidobranchia Styelidae MT232